Correlation of biological properties with glucan-binding protein B expression profile in Streptococcus mutans clinical isolates.

OBJECTIVE: Streptococcus mutans is known to be a primary causative agent of dental caries and its surface proteins have been investigated to specify their association with its virulence. Amongst those, 4 glucan-binding proteins (Gbps) are considered to be important factors due to their glucan-binding properties, of which GbpB has been shown to participate in cell-wall construction and cell separation. DESIGN: We examined clinical isolates of S. mutans collected from the oral cavities of Japanese and Finnish subjects, and focused on the association of their GbpB expression profiles and biological properties related to virulence. RESULTS: Western blot analysis of GbpB expression by the isolates revealed a variety of patterns. Strains that showed single and multiple bands were used to designate S and M type strains, respectively, whilst those with no GbpB expression were classified as N type. The distribution of GbpB expression patterns was shown to be quite different between the Japanese and Finnish isolates. Furthermore, the chain length and doubling time of the N type in both populations were significantly longer than those of the other types. CONCLUSION: Our results suggest variations in S. mutans GbpB expression patterns, which may have relationships with the virulence of S. mutans.

Molecular characterization of Streptococcus mutans strains containing the cnm gene encoding a collagen-binding adhesin.

OBJECTIVE: Streptococcus mutans, known to be a major pathogen of dental caries, is also considered to cause infective endocarditis. Its 120-kDa Cnm protein binds to type I collagen, which may be a potential virulence factor. In this study, we characterized S. mutans clinical strains focusing on the cnm gene encoding Cnm. DESIGN: A total of 528 S. mutans strains isolated from Japanese, Finnish, and Thai subjects were investigated. Using molecular techniques, the distribution frequency of cnm-positive strains and location of the inserted cnm were analyzed. Furthermore, isogenic mutant strains were constructed by inactivation of the cnm gene, then their biological properties of collagen-binding and glucan-binding were evaluated. Southern hybridization of the genes encoding glucan-binding proteins was also performed. RESULTS: The distribution frequency of cnm-positive strains from Thai subjects was 12%, similar to that previously reported for Japanese and Finnish subjects. Furthermore, the location of insertion of cnm was the same in all cnm-positive clinical isolates. As for the cnm-inactivated mutant strains constructed from 28 clinical isolates, their collagen-binding activity was negligible. In addition, glucan-binding activity in the cnm-positive clinical isolates was significantly reduced and corresponded to a lack of gbpA encoding glucan-binding protein A. CONCLUSIONS: Our results indicate that strains with cnm genes, the most crucial factor for the collagen-binding property of S. mutans, are detectable at similar frequencies over several different geographic locations. In addition, the common properties of these strains are a high level of collagen-binding activity and tendency for a low level of glucan-binding activity.

Protein antigen in serotype k Streptococcus mutans clinical isolates.

Streptococcus mutans, a major pathogen of dental caries and infective endocarditis, is classified into serotypes c, e, f, and k, with serotype k strains recently reported to be frequently detected in persons with infective endocarditis. Thus, we hypothesized that common properties associated with infective endocarditis are present in those strains. Fifty-six oral S. mutans strains, including 11 serotype k strains, were analyzed. Western blotting analysis revealed expression of the 3 types of glucosyltransferases in all strains, while expression of the approximately 190-kDa cell-surface protein (PA) was absent in 12 strains, among which the prevalence of serotype k (7/12) was significantly high. Furthermore, cellular hydrophobicity and phagocytosis susceptibility were lower in the group of serotype k strains. These results indicate that the absence of PA expression, low cellular hydrophobicity, and phagocytosis susceptibility are common bacterial properties associated with serotype k strains, which may be associated with virulence for infective endocarditis.

Site-specific oral colonization of mutans streptococci detected by arbitrarily primed PCR fingerprinting.

The clonal diversity and colonization pattern of mutans streptococci within an individual were studied by using a total of 50 tooth site samples obtained from seven 3- to 7-year-old children. Five children contributed saliva samples. From the 7 subjects, 90, 74, 81, 53, 83, 103, and 114 isolates (a total of 598) representing Streptococcus mutans and Streptococcus sobrinus were included for arbitrarily primed polymerase chain reaction (AP-PCR) fingerprinting. The children harbored one to four different AP-PCR types of mutans streptococci. In 3 of the 7 children, the distribution of AP-PCR types of S. mutans differed depending on tooth site (chi-square test for heterogeneity, p<0.001), suggesting that mutans streptococcal clones may selectively colonize specific hard-tissue sites.

Mutacin production by Streptococcus mutans may promote transmission of bacteria from mother to child.

The production of bacteriocin-like inhibitory substances, mutacins, by mutans streptococci varies among isolates. To find if the degree of mutacin activity of an isolate was related to its transmission between mother and her child, 19 mothers and their 18-month- to 3-year-old children were sampled for their oral mutans streptococci. In addition, the stability of mutacin activity was studied with isolates from the mothers and with isolates from five unrelated 5-year-old children in 5- to 7-year follow-up studies. A total of 145 oral mutans streptococcal isolates were serotyped by immunodiffusion, ribotyped, and mutacin typed by the stab culture technique. Mutacin was produced by 88% of the strains against more than 1 of the 14 indicator strains, representing mutans streptococci, Streptococcus sanguis, Streptococcus salivarius, Streptococcus oralis, Streptococcus gordonii, and Streptococcus pyogenes. Streptococcus mutans isolates showed more inhibitory activity than did Streptococcus sobrinus isolates. Identical ribotypes had similar mutacin activity profiles within a subject, initially and in the follow-up studies, in all but two cases. The mothers harbored a total of 37 different mutans streptococcal ribotypes. Six children were negative for mutans streptococci. Transmission was probable in 9 of 20 mother-child pairs on the basis of the presence of identical strains, as determined by ribotyping and bacteriocin (mutacin) typing. S. mutans strains shared between a mother and her child showed a broader spectrum of inhibitory activity than did nontransmitted strains. In conclusion, the mutacin activity of clinical isolates is reasonably stable, and this virulence factor seems to be of clinical importance in early colonization by S. mutans.

Production of glucosyltransferases by clinical mutans streptococcal isolates as determined by semiquantitative cross-dot assay.

Forty-four clinical isolates of mutans streptococci were examined by a semiquantitative cross-dot assay for in vitro production of glucosyltransferases GTF-I, GTF-SI and GTF-S of Streptococcus mutans, and GTF-I of Strep. sobrinus, using monospecific antibodies. The isolates were obtained from 12 1.5- to 3-year old children, six caries-active and six caries-free, and from their mothers. The isolates were selected originally from 243 isolates and they represented 35 genetically distinct types as analysed by serotyping and ribotyping. 27 isolates were of serotype c, nine of serotype e and eight of serotype g. Mother child pairs shared nine ribotypes, suggesting vertical transmission. The results showed that, when cultured in Todd-Hewitt broth supplemented with 1% glucose, all Strep. mutans isolates produced GTF-I and GTF-S and all except two produced GTF-SI of Strep. mutans. All Strep. sobrinus isolates produced GTF-I of Strep. sobrinus. The Strep. mutans GTF-I, GTF-SI and GTF-S production of isolates exhibiting a different ribotype showed variability. The variability of GTF-SI and GTF-S production was less pronounced for serotype e isolates. The GTF-I production by Strep. sobrinus isolates did not vary. Transmitted strains produced the same levels of GTFs as strains that were distinct (not transmitted). Strep. mutans isolates of caries-active children produced the same levels of GTF-I and GTF-S, but tended to produce lower levels of GTF-SI than isolates of caries-free children. In conclusion, the results suggested that Strep. mutans isolates exhibiting a different ribotype often had differences in production of GTFs. However, no clear superiority of the high-producer over the low-producer strains was found in regard to their colonization or caries promotion in young children.

Oral colonization by more than one clonal type of mutans streptococcus in children with nursing-bottle dental caries.

By ribotyping the genetic diversity of mutans streptococci in six 1.5-3-yr-old children with nursing-bottle caries and in six caries-free, age-matched children and in their mothers was examined. The proportion of mutans streptococci in the dental plaque of the children and their levels in the saliva of the mothers were also examined. For ribotyping, chromosomal DNA of isolates obtained from the plaque of the children (3-12 isolates per child) and from the saliva of the mothers (4-13 isolates per mother) was digested with restriction endonuclease HindIII. The DNA fragments were hybridized to the plasmid pKK3535 which contains the rRNA operon of the Escherichia coli chromosome. The results showed that children with nursing-bottle caries exposed to frequent consumption of sucrose had a high proportion of mutans streptococci in plaque and four of them were colonized with more than one ribotype, whereas caries-free children had a low proportion of mutans streptococci in plaque and only one of them harboured more than one ribotype. Mothers of children with nursing bottle caries had similar levels and numbers of ribotypes of mutans streptococci in saliva as the mothers of the caries-free children. In both child groups, mothers were probably the main source of infection with mutans streptococci. Thus, children with nursing-bottle caries were not only heavily infected with mutans streptococci but also often colonized with more than one clonal type. In the child's acquisition of such clones, frequent sugar consumption may have an important role.

Chlorhexidine susceptibilities of mutans streptococcal serotypes and ribotypes.

The susceptibilities of 379 clinical mutans streptococcal isolates to chlorhexidine (CHX) were tested by agar dilution according to the standards of the National Committee for Clinical Laboratory Standards. Isolates were obtained from saliva samples of 34 young mothers who had high or moderate salivary levels of mutans streptococci at baseline. Samples were collected on three occasions, before childbirth, when each child was 6 months old, and 1 year later. Of these isolates, 50% were inhibited at 1 microgram of CHX per ml, 90% were inhibited at 2.0 micrograms/ml, and all were inhibited at 4.0 micrograms/ml. The MICs for Streptococcus mutans isolates (serotypes c, e, and f) were lower than those for Streptococcus sobrinus isolates (serotypes d and g). In some subjects, the MICs for isolates of the same serotype were different. This phenomenon was studied by ribotyping isolates (n = 45) from selected subjects (n = 7). It was found that if there were intraindividual differences in the MICs for isolates of the same serotype, then the ribotypes of these isolates were different. In order to decrease the mutans streptococcal infection risk for children, 24 mothers (test group) brushed their teeth periodically with a gel that contained 0.3% CHX digluconate and 0.2% NaF, pH 5.8, between the second and third sampling occasions. The gel was used twice a day for the first 10 days of each month. Development of resistant strains during CHX-NaF gel use was not detected. The serotype distribution of isolates from the test group after 1 year of periodic CHX-NaF gel use did not differ from that at baseline. Periodic CHX-NaF gel brushing did not lead to lower salivary mutans streptococcal counts.

Effects of a chlorhexidine-fluoride-strontium rinsing program on caries, gingivitis and some salivary bacteria among Finnish schoolchildren.

In order to find out if it is possible to prevent caries and gingivitis by periodical use of chlorhexidine-fluoride mouthrinses with or without strontium, and to find out what effects they have on salivary mutans streptococci and lactobacilli counts, a total of 243 schoolchildren aged 11 yr with high DMFS scores were randomly divided into four groups. One group (C) served as a basic control. Subjects in the second group (CXF) rinsed their mouths twice a day every third week with a rinsing solution containing 0.05% chlorhexidine gluconate and 0.04% NaF. In the third group (CXFS) the rinsing solution contained 500 ppm Sr during the first and second year and 15 ppm during the last 6 months, in addition to chlorhexidine and fluoride. In the fourth group (CX) the solution contained only 0.05% chlorhexidine gluconate. All the rinsing solutions had pH 5.8 buffered with succinic acid-NaOH buffer. After 2 yr and 9 months, the mean DMFS (SD) increments in the C, CXF, CXFS, and CX groups were 3.8 (5.7), 2.5 (3.2), 3.5 (4.8), and 3.4 (5.5), respectively. The percentage of subjects with bleeding gingival units had decreased from initial to final values as follows: C, 81-38; CXF, 88-42; CXFS, 89-56; CX, 89-37. The number of lactobacilli and mutans streptococci in saliva remained virtually unchanged throughout the study. For caries increment and gingival bleeding, the differences between groups were not statistically significant. The chlorhexidine-fluoride combination tended to prevent caries, but the effect on gingival bleeding and salivary counts of mutans streptococci and lactobacilli was negligible.

Salivary caries-related tests as predictors of future caries increment in teenagers. A three-year longitudinal study.

The aim of the study was to evaluate the capability of caries-related salivary tests and a test based on past caries experience (baseline DFS) to select persons at high risk for caries. The subjects (n = 122) were 12-17 years old at the beginning of the study. Caries was registered and salivary samples were taken annually during the 3-study period. The 3-year caries increment was positively correlated to the baseline DFS (r = 0.46, p less than 0.001), salivary level of mutans streptococci (r = 0.30, p less than 0.001) and lactobacilli (r = 0.30, p less than 0.001), and combined level of mutans streptococci and lactobacilli (r = 0.39, p less than 0.001) and negatively correlated to the buffering capacity of saliva (r = -0.22, p less than 0.05). Tests based on either past caries experience or mutans streptococci or lactobacilli levels alone were not efficient in selecting persons at high risk for caries. Among the tests, DFS was the most sensitive and specific. A combination of either microbial test and DFS was more efficient to select persons at risk than various alternatives alone. The sensitivity was 84% and the specificity 62% for the combination of lactobacilli test and DFS and 71% and 79% respectively for the combination of mutans streptococci and DFS. In the former combination the positive prediction value was 43% and in the latter 56%.

Streptococcus mutans, not detected?

The growth of Streptococcus mutans was followed for three years in 24 healthy 12-17 year-old children, who were selected for the study on the basis of undetectable levels of salivary S. mutans. The saliva samples were cultured by a dip-slide method based on mitis-salivarius agar supplemented with sucrose and bacitracin. At the final occasion, additional examination of plaque and saliva of 10 children using TSY20B agar was performed. During the 3-year study period, S. mutans was found in saliva samples from 15 children on one or more of the 4 test occasions, suggesting that most 'S. mutans negative individuals' were those in whom S. mutans could not be temporarily detected. The additional examination of plaque did not increase the number of 'S. mutans-positive individuals'.

Caries-related microbiological findings in a group of teenagers and their parents.

The levels of salivary Streptococcus mutans and lactobacilli and caries experience were studied in 113 teenagers and 163 adults. The study population consisted of 82 mother-child and 73 father-child pairs. The number of decayed, missing, and filled tooth surfaces (DMFS) was eightfold higher in adults than in teenagers (56.4 +/- 22.8 vs. 7.3 +/- 6.7). The percentage distribution of the level of salivary S. mutans and lactobacilli was approximately the same in both groups. The mean number of DMFS increased with increasing levels of salivary S. mutans and lactobacilli, the correlation being highly significant both in teenagers and adults. There was a significant correlation of the DMFS indices in the mother-child pairs (r = 0.364), but the correlation was not significant in the father-child pairs (r = 0.138). The salivary level of S. mutans was higher in the children of mothers with high DMFS values compared to the children of mothers with low DMFS values.

Slide-scoring method for estimation of Streptococcus mutans levels in saliva.

A method was developed to facilitate the estimation of S. mutans levels in saliva. Paraffin-stimulated saliva was poured on a special slide coated with mitis-salivarius-sucrose agar. Two discs containing bacitracin (5 micrograms) were placed on the inoculated slide and the growth density of S. mutans around the bacitracin discs was scored after incubation in candle jars at 37 degrees C for 48 h. The obtained score values correlated well with the numbers of CFU of S. mutans per 1 ml of saliva estimated by a conventional method using mitis-salivarius-bacitracin agar. The experimental method was further tested by incubating the slides in an atmosphere created by CO2-generating tables that were placed into the cover tubes of the slides. These score values were similar to those after conventional incubation. The method is suggested for epidemiologic studies and in selecting persons at high risk for caries and in controlling the effectiveness of prophylactic measures of these patients.